Composition and Biological Properties of Lipopolysaccharides Isolated from Schizothrix Calcicola (Ag.) Gomont (Cyanobacteria) GEORG KELETI,'* JAN L
Total Page：16
File Type：pdf, Size：1020Kb
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1979, p. 471-477 Vol. 38, No. 3 0099-2240/79/09-0471/07$02.00/0 Composition and Biological Properties of Lipopolysaccharides Isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria) GEORG KELETI,'* JAN L. SYKORA,' EDWIN C. LIPPY,2 AND MAURICE A. SHAPIRO' Graduate School ofPublic Health, University ofPittsburgh, Pittsburgh, Pennsylvania 15261,' and U.S. Environmental Protection Agency, Health Effects Research Laboratory, Cincinnati, Ohio 452682 Received for publication 15 March 1979 The most common cyanobacterium contaminating drinking water systems in southwestern Pennsylvania is Schizothrix calcicola. Lipopolysaccharides (LPS) were isolated from this species by hot phenol-water extraction. The polysaccha- ride moiety was composed of glucosamine, galactose, glucose, mannose, xylose, and rhamnose. The lipid A part contained beta-hydroxylauric, myristic, penta- decanoic, palmitic, beta-hydroxypalmitic, stearic, oleic, and linoleic acids. In contrast to many LPS isolated from Enterobacteriaceae, the dominant compo- nent was not beta-hydroxymyristic but beta-hydroxypalnitic acid. The LPS induced Limulus lysate gelation and Schwartzman reaction but was nontoxic to mice. The identity of LPS was verified by alkali and lysozyme treatment. The results suggest that S. calcicola is one of the principal sources of endotoxins in water systems using open finished-water reservoirs. A water-bome outbreak of gastroenteritis af- [LPS]) have been isolated from six species of fected approximately 5,000 persons in Sewickley, cyanobacteria (2, 17, 29, 30). LPS are constitu- Pa., during August 1975. Extensive microbiolog- ents of the outer cell wall of some microorga- ical and chemical analyses of specimens ob- nisms, predominantly gram-negative bacteria. tained from patients and of water samples failed These macromolecular substances are composed to identify a causative agent. However, investi- ofthree regions. The first region is the 0-specific gation of the Sewickley water system revealed polysaccharide consisting of repeating oligosac- an accumulation of Schizothrix calcicola (Cy- charide units, different in various species of bac- anobacteria = Cyanophyta) in the open fin- teria. The second region is the basal core oligo- ished-water reservoirs (15). Even 1 month after saccharide, and the third region is the hydro- the outbreak, the water in the reservoir with the phobic lipid part, the lipid A. Many gram-nega- longest detention time was still contaminated by tive bacterial mutants possess only glycolipids, this species (400,000 cells ml-'). and the 0-specific polysaccharide is absent. Toxic algae and cyanobacteria have been rec- Sometimes even a part of the core oligosaccha- ognized since antiquity. They are found in di- ride is lacking. verse aquatic environments and have been im- The LPS derived from blue-green organisms plicated in poisoning and death of fish, wildlife, are generally identical to those occurring in domestic animals, and humans (18). Many ob- gram-negative bacteria. However, the available served episodes were caused by ingestion of wa- though limited qualitative data on the compo- ter from reservoirs, lakes, and ponds contamined sition of cyanobacterial LPS have already re- by cyanobacteria. Since these organisms are vealed a diversity in their chemical composition widely distributed, it is not surprising that cy- and biological characteristics. anobacteria were associated with several water- In this report we describe the composition and borne outbreaks of gastroenteritis in the United biological properties of LPS isolated from S. States (25, 26), the Philippines (3), and India (7). calcicola. S. calcicola is not usually listed among the toxic cyanobacteria, and there is only one publication MATERIALS AND METHODS suggesting that this species may form toxic Enumeration of algae and cyanobacteria. Al- strains (1). gae and cyanobacteria were counted in samples col- In addition to toxins (anatoxin, saxitoxin, toxic lected from five different water systems in the Pitts- polypeptides), macromolecular substances char- burgh, Pa., area. All of the water treatment plants acterized as endotoxins (lipopolysaccharides selected for sampling stored finished water in open 471 472 KELETI ET AL. APPL. ENVIRON. MICROBIOL. reservoirs which varied in size from 0.5 to 177.5 million eliminated by enzymatic hydrolysis with a beta-glu- gallons. The samples were collected at weekly inter- cosidase as recommended by Volk (27). Cellulase (2.5 vals during late spring and early summer (April-June mg ml-'; Sigma Chemical Co.) was dissolved in water 1977) from the treated water in each plant, open and centrifuged at 37,000 x g for 15 min, and the reservoir(s), and distribution system(s). Algae were supernatant was passed through a 0.45-nm-pore size preserved with Lugol solution, concentrated by sedi- membrane filter (Millipore Corp.). The crude LPS was mentation, and enumerated in a Palmer-Maloney nan- suspended at 5 mg ml-' in 0.05 M citrate buffer (pH noplankton cell. The results were expressed as number 4), and a final concentration of 0.25 mg of enzyme was of cells per milliliter. added per 1 mg of LPS. After 10 h of treatment at Cultures of S. calcicola. A brown strain of S. room temperature, a second equal amount of cellulase calcicola identical to that occurring in drinking water was added, and the enzymatic hydrolysis was contin- was isolated from Mosquito Creek Reservoir, near ued overnight. Afterwards the mixture was dialyzed Youngstown, Ohio. It was identified by using cell for 48 h against tap water, concentrated in a rotary morphology, color, and size and by comparison with evaporator, and freeze-dried. To remove most of the the organisms obtained from Sewickley Reservoir no. nucleic acids, an aqueous solution (1 to 2%) of the 4. The identification was confirmed by Francis Drouet freeze-dried, crude LPS was further purified by re- of the Academy of Natural Sciences of Philadelphia. peated (three times) centrifugation at 105,000 x g in S. calcicola was mass cultured in 9-liter bottles using a Spinco ultracentrifuge for 4 h. The sediment was Allen medium (9) kept at 24°C, aerated with com- resuspended in distilled water and centrifuged for 10 pressed air at a rate of 100 to 250 ml/min, and illumi- min at 3,000 rpm. The supernatant, representing the nated by white fluorescent light adjusted to a 16-h purified LPS, was lyophilized. cycle. Each bottle was inoculated with 200 ml of pure, The phenol phase of the extracted cells was sub- aerated starter culture. For comparison, several mass jected to the same procedure as the aqueous phase, cultures of S. calcicola were prepared in a 14-liter and only a negligible contamination by LPS was iso- Microferm MF-114 fermentor (New Brunswick Sci- lated from this fraction. entific Corp.) with aeration and illumination and Analytical procedures. The neutral and amino equipped with constant temperature controls. For sugars were released from the LPS by using hydrolysis endotoxin isolation, 14-day-old cultures were har- with 1 N H2SO4 and 4 N HCl in a boiling-water bath vested at 3,450 rpm using a flow-through centrifuge for 4 and 12 h, respectively. After neutralization and (Gyrostester, DeLaval Separator Co.), washed three subsequent centrifugation at 2,000 rpm for 10 min at times, and lyophilized. 4°C, the supernatant was cooled and dried in a vacuum The heterotrophic bacterial contamination of S. desiccator equipped with P205 and, in the case of calcicola was determined by plating serial dilutions amino sugars, also with NaOH pellets. Final analysis (0.85% saline) of the cultures on Trypticase soy agar of the released sugars was performed using descending (BBL Microbiological Systems) in duplicate. The paper chromatography with proper sugar standards plates were incubated for 72 h at 20 and 35°C. Because on Whatman no. 1 paper using n-butanol-pyridine- the majority ofthe bacteria contaminating S. calcicola water (6:4:3, vol/vol/vol) (12). were from the family Enterobacteriaceae, a laboratory For fatty acid analysis of the lipid A part, the LPS strain of Salmonella typhimurium was selected as the was hydrolyzed with 4 N HCl in a boiling-water bath biomass indicator. The bacterial biomass was calcu- for 5 h, followed by chloroform extraction repeated lated utilizing a 24-h culture of this strain in soybean three times. The combined chloroform extract was casein digest broth as a standard. A series of dilutions dried in a vacuum rotary evaporator, and the residue in physiological saline containing this organism was was dissolved in ethyl ether and dried in a sand bath plated on Trypticase soy agar medium, and the num- at 37°C. The sample was methylated with 0.5 ml of ber of colonies of S. typhimurium present in the cul- BF3 methanol (14%, wt/vol) in a tightly stoppered ture was determined. One liter of the same culture was flask, placed in a boiling-water bath for 2 min, and, centrifuged, washed three times, and lyophilized, and after cooling, extracted twice with petroleum ether. the dry weight of bacteria was established. Based on The combined petroleum ether phases were dried at this standard, the average count of heterotrophic bac- 60°C in a sand bath and dissolved in ethyl acetate teria contaminating S. calcicola cultures constituted before chromatographic and mass spectrometric anal- only 0.04% of the total biomass. ysis. Isolation and purification ofLPS. The harvested The methyl esters of the fatty acids were examined cells were washed three times with sterile, distilled on a Packard series 7400 gas chromatograph (Packard water. Each washing in a Sorvall (RC-2B) centrifuge Instrument Co., Downers Grove, Ill.) using a 3% OV- lasted 25 min at 5,500 rpm and 4°C. The resultant 17 glass column (2 mm by 10 feet [ca. 3.05 ni]). The sediment was uniformly suspended in distilled water analyses were programmed from 100 to 300°C at 7°C/ and freeze-dried.